SUMO proteins are a conserved family of ubiquitin-related proteins that become conjugated to substrates in a manner similar to ubiquitin. Fission and budding yeast each contain a single SUMO family protein. These proteins have been implicated in the regulation of the cell cycle in both organisms. There are three human SUMO paralogues: SUMO-1 is about 45% identical to SUMO-2 and SUMO-3, which are 96% identical to each other. The conjugation pathway for all paralogues is similar to the ubiquitin conjugation pathway: SUMO proteins must be processed to yield a C-terminal di-glycine motif. After processing, the first step in the SUMO conjugation pathway is the ATP-dependent formation of a thioester bond between SUMO proteins and their activating (E1) enzyme. The second step is the formation of a thioester bond between SUMO proteins and their conjugating (E2) enzyme, Ubc9. In the last step, an isopeptide bond is formed between SUMO proteins and substrates through the cooperative action of Ubc9 and protein ligases (E3). An important question about SUMO proteins concerns the roles of individual SUMO paralogues within vertebrate cells. It is currently unclear whether SUMO-1, -2 and -3 function in ways that are unique, redundant or antagonistic. Moreover, all three paralogues share common E1 and E2 enzymes, while the specificity of SUMO ligases and proteases is not well understood. It has been difficult to address this question experimentally in the past, because superphysiological levels of individual SUMO proteins can cause a loss of paralogue specificity, and because the dynamics and localizations of these proteins can only be imprecisely estimated within fixed cells. To address the dynamic properties of SUMO paralogues, we have developed stable HeLa-derived cell lines that express biofluorescent SUMO chimeras at levels comparable to the endogenous proteins. Through live imaging and photobleaching studies, we have found that SUMO-1 differs from SUMO-2 and SUMO-3 in both it localization and its dynamics throughout the cell cycle. Additionally, we found significant differences between SUMO-1 dynamics in different subnuclear compartments. Together, our findings demonstrate that mammalian SUMO paralogues show discrete temporal and spatial patterns of utilization throughout the cell cycle, arguing that they are functionally distinct and specifically regulated in vivo. In addition to experiments examining the role of SUMO-1 conjugation in the regulation of RanGAP1, we have sought to identify conjugation targets whose modification is cell cycle-dependent. We have found that Topoisomerase-II is modified exclusively by SUMO-2/3 during mitosis in Xenopus egg extracts; this modification is maximal in metaphase, followed by rapid deconjugation during anaphase. The differential extraction properties of modified and unmodified Topoisomerase-II suggest that SUMO-2/3 conjugation may mobilize Topoisomerase-II from mitotic chromatin in a manner that is important for chromosome segregation. Together, our findings indicate that SUMO-2/3 conjugation of Topoisomerase-II is important for remodeling of mitotic chromosomes at the metaphase-anaphase transition, and that failure of such remodeling could be expected to cause high levels of chromosome mis-segregation in vivo. We have identified the SUMO ligase that is responsible for the mitotic conjugation of Topoisomerase-II, and we are currently investigating how it is regulated by phosphorylation and association to chromatin. We have also been systematically examining the properties and behavior of SUMO ligases (PIAS/Siz family) and SUMO protease (SENPs). We find that PIAS family members show similar patterns of localization within interphase nuclei, but also show subtle differences in their localization on mitotic chromosomes. We have identified domains within one of the PIAS proteins (PIASy) that are responsible for its targeting to interphase nuclei and to chromosomes. Different SENP family members are localized to nucleoli, nucleoplasm and to the nuclear envelope. We are currently pursuing identification of the interactions responsible for such targeting, and investigating the consequences of mis-targeting these enzymes.